While many anti-retroviral inhibitors have been developed against HIV-1 infection and replication, the effectiveness of these drugs is severely hindered by the rapid development of resistant viral strains. Both the low fidelity of proviral DNA synthesis by reverse transcriptase (RT) and the high rate of recombination during reverse transcription have been implicated in this variation. The overall goal of this work is to understand the fundamental mechanisms of HIV-1 reverse transcriptase (RT) fidelity and drug resistance. From a mechanistic standpoint, this will contribute greatly to our understanding of enzyme specificity and protein dynamics. From a medical standpoint, understanding the mechanisms surrounding the fidelity of HIV-1 DNA synthesis are essential to our understanding of the development of drug resistance to any HIV-1 target. In addition, since HIV-1 RT shares structural and kinetic similarities with a variety of other DNA and RNA polymerases, these studies will serve as a model for understanding the mechanism and protein dynamics of a wide range of replicating enzymes. The important enzyme-substrate interactions required to maintain the fidelity of nucleotide incorporation during DNA synthesis are largely unknown. To study this phenomenon at the molecular level, we will utilize steady-state and presteady-state kinetic methods in combination with spectrofluorometric techniques to examine mutants of HIV-1 RT known to have both drug resistance and altered fidelity characteristics. The proposed experiments will allow us to determine which step along the complex polymerase reaction pathway is effected by a given mutation. In addition, we have developed methodologies that allow the screening of mutant HIV-1 RT libraries for additional protein variants displaying altered catalytic characteristics resulting in higher or lower fidelity of DNA synthesis. Aided by the crystal structures of HIV-1 RT, we will identify specific amino acid residues/structural domains of HIV-1 RT that are involved in nucleotide binding and recognition, and through rigorous kinetic analysis we will be able to understand what portion of the enzyme reaction mechanism is influenced by the mutagenic changes. Methods for examining important protein conformational changes involved in substrate nucleotide discrimination are presented that will help define both the kinetic/thermodynamic and structural features of polymerase fidelity. Understanding the kinetic, molecular and catalytic mechanism of these mutants will help establish a picture of HIV-1 RT that correlates enzyme structure, substrate recognition, catalysis and nucleoside analog drug resistance.